Title: Meta-transcriptomics and the evolutionary biology of RNA viruses
Mang Shi ...
2 January 2018
Highlights
- Meta-transcriptomics (bulk RNA-Seq) is a powerful new way to characterise viromes.
- Meta-transcriptomic data are changing our understanding of virus evolution.
- Invertebrates harbor an enormous phylogenetic and genomic diversity of RNA viruses.
- Present sampling schemes have only revealed a miniscule fraction of the virosphere.
- The new wealth of virus genomic data presents a major challenge to classification.
Abstract
Metagenomics is transforming the study of virus evolution, allowing the full assemblage of virus genomes within a host sample to be determined rapidly and cheaply. The genomic analysis of complete transcriptomes, so-called meta-transcriptomics, is providing a particularly rich source of data on the global diversity of RNA viruses and their evolutionary history. Herein we review some of the insights that meta-transcriptomics has provided on the fundamental patterns and processes of virus evolution, with a focus on the recent discovery of a multitude of novel invertebrate viruses. In particular, meta-transcriptomics shows that the RNA virus world is more fluid than previously realized, with relatively frequent changes in genome length and structure. As well as having a transformative impact on studies of virus evolution, meta-transcriptomics present major new challenges for virus classification, with the greater sampling of host taxa now filling many of the gaps on virus phylogenies that were previously used to define taxonomic groups. Given that most viruses in the future will likely be characterized using metagenomics approaches, and that we have evidently only sampled a tiny fraction of the total virosphere, we suggest that proposals for virus classification pay careful attention to the wonders unearthed in this new age of virus discovery.
Outline
1. Introduction: virology in the age of metagenomics
2. Overview of meta-transcriptomics
3. Implications of meta-transcriptomics for virus evolution
3.1 A new view of virus diversity
3.2 Linking the vertebrate and invertebrate worlds
3.3 Cross-species transmission and emergence
3.4 The evolution of genome structures
4. Implications of meta-transcriptomics for virus taxonomy
5. Conclusions and future directions
正文摘錄：
1.Introduction:
第一段：之前一直是研究DNA病毒，後來開始關注RNA病毒
Although viruses are the most abundant source of nucleic acid on earth, with every species of cellular life likely harboring multiple viruses, until recently most studies of virus biodiversity and evolution were of limited scope, with a strong focus on aquatic environments and prokaryotic DNA viruses
第二段：引出元轉錄組學
As the transcriptome data generated by RNA-Seq is able to provide an unbiased and likely comprehensive view of all the viruses present within a host sample – that is, their complete virome – it can also be thought of as ‘meta-transcriptomics’.
元轉錄組學的作用，進化，鑑定新型微生物病原體，可能會用於常規的微生物學診斷
Aside from its evolutionary utility which we will discuss in more detail below, meta-transcriptomics allows the identification of novel microbial pathogens
meta-transcriptomics may eventually be used for routine microbiological diagnostics.
第三段：本review的目標：回顧我們認爲元轉錄組學告訴我們的有關病毒多樣性，進化和分類的內容，併爲該領域的未來工作提供一些建議。
we will review what, in our opinion, meta-transcriptomics has told us about virus diversity, evolution and taxonomy, and provide some suggestions for future work in this area.
2.Overview of meta-transcriptomics
先回顧了之前病毒研究的方法
The most robust, although costly, method of virus discovery is through a coupling of metagenomics and high-throughput sequencing technology.
引出metagenomics（成本較高）最有效
宏轉錄組學是宏基因組學的一種方法
Among the various metagenomics approaches are available, meta-transcriptomics has recently come to the fore.
宏轉錄組學與viral particle enrichment方法的對比，有很多優勢
Compared to metagenomics protocols that involve viral particle enrichment (reviewed in Kumar et al., 2017), this method is far simpler yet still achieves a high level of sensitivity, generality, and efficiency for virus discovery (Fig. 1).
Previous methodologies were often based on removing as much nucleic acid outside viral particles as possible by filtering, centrifugation, lysis, and nuclease treatment, although this seldom results in a complete depletion of host RNA (Firth and Lipkin, 2013, Mokili et al., 2012). In contrast, in meta-transcriptomics total RNA (i.e. the transcriptome) is directly extracted from untreated homogenates and used for library preparation without filtering and nuclease digestion steps.
meta-transcriptomics 的另一個優勢：提供了一種量化樣品中每種病毒的簡便方法。
Another benefit of meta-transcriptomics is that it provides a ready way to quantify each virus present in a sample. Specifically, the percentage of reads that map to a particular virus genome is a good indication of how abundant any virus is, especially in the context of conserved host genes (Shi et al., 2016a, Shi et al., 2017).
丰度信息可以和疾病相關聯
In turn, abundance level can provide important pointers to disease associations, whether viruses are segmented (such that genomic components have similar or different expression levels), and help identify those viruses that are in fact derived from other eukaryotic organisms present in the host sampled, such as in undigested food or prey, gut micro flora, and parasites, or simply contamination (and the greater the virus abundance, the more likely that active viral infection has occurred in the host under consideration).
In addition, compared to genomic nucleic acid, the transcriptome comprises compact information that is more balanced across domains of life, thereby preventing the over-dominance of genetic information from large cellular organisms.
3. Implications of meta-transcriptomics for virus evolution
3.1 A new view of virus diversity
現在的轉錄組學研究表明了我們對RNA病毒的多樣性瞭解才一點點（RNA病毒也是）
Those meta-transcriptomic studies undertaken to date have transformed our understanding of the extent and nature of viral biodiversity, making it abundantly clear that we have only sampled a tiny fraction of RNA virus biodiversity (as will also be true of DNA viruses).
it is possible that such highly biased sampling has distorted our view of virus evolution. 高度偏向的採樣可能會扭曲我們對病毒進化的看法。
實際上，最近估計真核病毒球體約99.995％仍未被發現或未分類（Geoghegan和Holmes，2017年）。 因此，現實是，我們對病毒多樣性和進化以及分類學的研究纔剛剛開始。
Indeed, it was recently estimated that approximately 99.995% of the eukaryotic virosphere remains undiscovered or unclassified (Geoghegan and Holmes, 2017). The reality, therefore, is that our study of virus diversity and evolution, and hence taxonomy, has only just begun.
元轉錄組學揭示的新的多樣性財富也表明，病毒世界比我們以前認爲的要緊密得多。很多未發現的可以合併爲更大的組。顯然，越來越多來自不同宿主的新描述的病毒將繼續填補系統發育多樣性（即病毒間系統發育中存在的長分支）的“空白”，從而更加可靠地描述了病毒的進化歷史。
The new wealth of diversity revealed by meta-transcriptomics also shows that the virus world is far more connected than we previously thought. New broad-scale RdRp phylogenies have shown that virus families, orders, floating genera, and undefined lineages can often be amalgamated into larger groups, such that they exhibit an evolutionary continuity (Shi et al., 2016a), in turn providing compelling evidence for their common origin (Koonin et al., 2015). It is obvious that the increasing number of newly described viruses from diverse hosts will continue to fill ‘gaps’ in phylogenetic diversity (i.e. the long branches present in inter-virus phylogenies) resulting in a more robust and stable depiction of virus evolutionary history.
3.2 Linking the vertebrate and invertebrate worlds
It is now clear that invertebrates carry a huge diversity of RNA viruses，
What is far less clear is how frequently this huge array of invertebrate viruses is associated with overt disease in their hosts and, if invertebrates are largely refractory to disease, how this is mediated.
脊椎動物病毒特異性
Clearly, the monophyletic nature of vertebrate-specific viruses implies that have had a long-term evolutionary association with vertebrate hosts.
脊椎動物和無脊椎動物擁有共同的祖先
Therefore, while it is tempting to conclude that most, if not all, families of vertebrate viruses will have their ultimate ancestry with invertebrates, particularly as so very few of the latter have been sampled, it would be wrong to think that this a forgone conclusion.
3.3 Cross-species transmission and emergence
元轉錄組提供了一種簡便的方法確定病毒的宿主範圍
Determining the host range of viruses is essential to understanding the process of cross-species transmission that underpins disease emergence. Meta-transcriptomic data provide a ready means to determine what viruses are present in which hosts and allows a simple measure of virus abundance.
元轉錄組學和系統發育學的結合還告訴我們，病毒進化是跨物種傳播和病毒-宿主共生之間的複雜相互作用，許多病毒羣的進化歷史反映了兩個過程的相互交織
The combination of meta-transcriptomics and phylogenetics has also told us that virus evolution is a complex interaction between cross-species transmission and virus-host co-divergence, with the evolutionary history of many virus groups reflecting an interweaving of both processes (Geoghegan et al., 2017).
跨物種傳播，RNA病毒進化的一種方式？
At the same time, however, it is clear that cross-species transmission has occurred frequently, even among phylogenetically divergent taxa, and is likely the dominant mode of RNA virus evolution
可能會破壞基於生物多樣性的預測下一個主要疾病大流行的病毒來源的嘗試
Finally, although meta-transcriptomics has profound implications for our understanding of virus evolution, it likely undermines biodiversity-based attempts to predict the virus source of the next major disease pandemic (Olival et al., 2017).
元轉錄組學告訴我們，自然界中存在着如此多的病毒，以至於僅僅通過多樣性抽樣就試圖確定最終將出現在新宿主中的病毒幾乎肯定是徒勞的。這在當前研究蝙蝠病毒的流行中很明顯。
Meta-transcriptomics tells us that there are so many viruses in nature that trying to establish which will ultimately appear in a new host from diversity sampling alone is almost certainly a futile exercise. This is apparent in the current vogue to study bat viruses. Since the emergence of SARS coronavirus in humans – a pathogen that has its ultimate ancestry in bats – sampling bat viruses as a means to determine which next might emerge in humans has received considerable attention (Smith and Wang, 2013). While these studies have made it clear that bats indeed harbor an enormous number of viruses (Anthony et al., 2017, Luis et al., 2013, Olival et al., 2017), at the same time they clearly show that the vast majority of these viruses have not jumped to humans.
研究疾病出現的真正目標應該是揭示遺傳和生態因素的結合，這些因素是成功跨物種傳播和出現的基礎
The true goal of studies of disease emergence should therefore be to reveal that combination of genetic and ecological factors that underpins successful cross-species transmission and emergence.
3.4 The evolution of genome structures
宏基因組學數據最重要的影響之一就是改變了我們對病毒基因組結構和引起它們的進化過程的理解
One of the most important impacts of metagenomic data has been to change our understanding of the structure of virus genomes and the evolutionary processes that have given rise to them.
一種新興的觀點以及研究新病毒產生的方法：重要的是使用新的大量的轉錄組數據來仔細確定基因重複和丟失，
Indeed, an emerging view is that RNA viruses experience as complex processes of genome evolution as in DNA organisms. To better determine the evolutionary processes that shape viral genome structures, and hence how new viruses are created, it is important to use the new wealth of meta-transcriptomic data to carefully determine the frequency, pattern and history of gene duplications and losses, lateral gene transfers, and genomic rearrangements; combined, these will provide a more complete picture of genome-scale evolutionary processes obtained.
元轉錄組學從根本上改變了之前的一種關於黃病毒研究的觀點
However, segmentation no longer appears to be a strong taxon defining trait, and a combination of segmented and unsegmented genomes has now been observed within families of RNA viruses.
也許是元轉錄組學的一個缺陷？存在大小限制原因的爭論
Despite such a data revolution, one key feature of RNA virus genomes that has held firm in the metagenomics revolution is an upper-limit on genome length of <35 kb, with ball python nidovirus exhibiting the largest RNA virus genome reported to date – at 33.5 kb (Stenglein et al., 2014).
4. Implications of meta-transcriptomics for virus taxonomy
將來表徵病毒的主要方法將是通過宏基因組學調查
Indeed, there is now a growing recognition that the primary way in which viruses will be characterized in the future will be through metagenomic surveys
因爲我們的採樣的極其不足，目前的分類必然是不完整的
一個更根本的問題是當前的分類方案是否可以承受宏基因組學數據的衝擊？
Most importantly, phylogenetic trees are only ever able to depict the relationship among those viruses that are present in the sample of viruses under study; as our sample is likely negligible, so our classification is necessarily incomplete. A more fundamental question is whether the current classification scheme can withstand the onslaught of metagenomic data? The proliferation of ‘family-like’ viruses revealed from meta-transcriptomic surveys amply highlights the scale of the challenge facing taxonomists.
5. Conclusions and future directions
進行迄今尚未檢查過的原核生物分類的無偏宏基因組學調查，然後進行能夠準確識別病毒並揭示其系統進化關係的新型生物信息學分析至關重要。
It is therefore of critical importance to perform unbiased metagenomics surveys of prokaryotic taxa that have not been examined to date, followed by novel bioinformatics analyses that are able to accurately identify viruses and reveal their phylogenetic relationships.
宏轉錄組學在病毒研究方面可以解決的問題包括：
Key questions for future research that can be addressed with the new wealth of meta-transcriptomic data include (i) determining the flow of viruses between host taxa and the processes that shape virus ecosystems; (ii) revealing the mechanisms of long-term virus macroevolution, particularly lineage birth and death, and (iii) revealing the mechanisms and evolutionary processes that structure viral genomes.